Research Interests Research in our laboratory is devoted to understanding the mechanisms by which entire regions of the genome are rendered inaccessible to transcription and recombination. We employ genetic analysis coupled with biochemical fractionation and reconstitution experiments to explore the issues of genome accessibility. Current Research Silencing of genomic domains requires a complex series of interactions between inactivation centers called silencers and numerous repressor proteins. The silencers recruit repressor protein complexes composed of the Sir proteins that interact with histones in nucleosomes to form a chromatin domain that is inaccessible and inert to various cellular processes. We are currently focused on the Sir proteins and their interactions with the histones to understand in molecular detail the mechanism by which these proteins effect silencing. We are presently working on 1) The biochemical and molecular characterization of the structure of the silent domain and its reconstitution in vitro 2) The mechanism by which the silent domain is restricted to a specific region of the genome and 3) The role of histone variants in silencing and cell cycle progression. We have been purifying Sir protein complexes and analyzing the composition of these complexes and their associated enzymatic activities to understand the mechanism by which these proteins function in silencing. We have also begun studies on the reconstitution of silenced chromatin using these purified complexes and histones in nucleosomes. These studies will provide an important index of our current understanding of transcriptional silencing depending on whether or not it is possible to reconstitute the silenced state, since mechanisms are rarely established by genetic means and usually require biochemical tests. In addition to these studies we are also interested in understanding the mechanism by which the silenced chromatin domains are restricted to specific regions along the DNA fiber. We have demonstrated that specific elements act as barriers to the continuous spread of the silenced chromatin and our results suggest that barrier activity may arise from an underlying competition between chromatin remodeling and silencing activities at the interface of euchromatin and heterochromatin. We are currently performing a genome wide screen for proteins that can block the spread of silenced chromatin in order to determine the general principles that underlie the mechanism of barrier function. Our recent genetic studies have led to the identification of a histone H2A variant called Htz1p that is required for silencing. We are currently interested in understanding the many roles of this protein in the cell. Classical molecular genetic and biochemical experiments are in progress to determine the domains of Htz1 involved in cell cycle progression, the response to drugs as well as the proteins that interact with Htz1 to function in the cell. Together, these studies will allow a better understanding of the process of silencing and yield new insight into the mechanism by which genes are silenced.